Biosynthesis and Genetic Encoding of Non-hydrolyzable Phosphoserine into Recombinant Proteins in Escherichia coli

While site-specific translational encoding of phosphoserine (pSer) into proteins in Escherichia coli via genetic code expansion (GCE) technologies has transformed our ability to study phospho-protein structure and function, recombinant phospho-proteins can be dephosphorylated during expression/purification, and their exposure to cellular-like environments such as cell lysates results in rapid reversion back to the non-phosphorylated form. To help overcome these challenges, we developed an efficient and scalable E. coli GCE expression system enabling site-specific incorporation of a non-hydrolyzable phosphoserine (nhpSer) mimic into proteins of interest. This nhpSer mimic, with the γ-oxygen of phosphoserine replaced by a methylene (CH2) group, is impervious to hydrolysis and recapitulates phosphoserine function even when phosphomimetics aspartate and glutamate do not. Key to this expression system is the co-expression of a Streptomyces biosynthetic pathway that converts the central metabolite phosphoenolpyruvate into non-hydrolyzable phosphoserine (nhpSer) amino acid, which provides a > 40-fold improvement in expression yields compared to media supplementation by increasing bioavailability of nhpSer and enables scalability of expressions. This “PermaPhos” expression system uses the E. coli BL21(DE3) ΔserC strain and three plasmids that express (i) the protein of interest, (ii) the GCE machinery for translational installation of nhpSer at UAG amber stop codons, and (iii) the Streptomyces nhpSer biosynthetic pathway. Successful expression requires efficient transformation of all three plasmids simultaneously into the expression host, and IPTG is used to induce expression of all components. Permanently phosphorylated proteins made in E. coli are particularly useful for discovering phosphorylation-dependent protein–protein interaction networks from cell lysates or transfected cells. Key features • Protocol builds on the nhpSer GCE system by Rogerson et al. (2015), but with a > 40-fold improvement in yields enabled by the nhpSer biosynthetic pathway. • Protein expression uses standard Terrific Broth (TB) media and requires three days to complete. • C-terminal purification tags on target protein are recommended to avoid co-purification of prematurely truncated protein with full-length nhpSer-containing protein. • Phos-tag gel electrophoresis provides a convenient method to confirm accurate nhpSer encoding, as it can distinguish between non-phosphorylated, pSer- and nhpSer-containing variants.

This protocol is used in: ACS Cent.Sci.(2023), DOI: 10.1021/acscentsci.3c00191While site-specific translational encoding of phosphoserine (pSer) into proteins in Escherichia coli via genetic code expansion (GCE) technologies has transformed our ability to study phospho-protein structure and function, recombinant phospho-proteins can be dephosphorylated during expression/purification, and their exposure to cellular-like environments such as cell lysates results in rapid reversion back to the non-phosphorylated form.To help overcome these challenges, we developed an efficient and scalable E. coli GCE expression system enabling site-specific incorporation of a non-hydrolyzable phosphoserine (nhpSer) mimic into proteins of interest.This nhpSer mimic, with the γ-oxygen of phosphoserine replaced by a methylene (CH2) group, is impervious to hydrolysis and recapitulates phosphoserine function even when phosphomimetics aspartate and glutamate do not.Key to this expression system is the co-expression of a Streptomyces biosynthetic pathway that converts the central metabolite phosphoenolpyruvate into non-hydrolyzable phosphoserine (nhpSer) amino acid, which provides a > 40-fold improvement in expression yields compared to media supplementation by increasing bioavailability of nhpSer and enables scalability of expressions.This "PermaPhos" expression system uses the E. coli BL21(DE3) ΔserC strain and three plasmids that express (i) the protein of interest, (ii) the GCE machinery for translational installation of nhpSer at UAG amber stop codons, and (iii) the Streptomyces nhpSer biosynthetic pathway.Successful expression requires efficient transformation of all three plasmids simultaneously into the expression host, and IPTG is used to induce expression of all components.Permanently phosphorylated proteins made in E. coli are particularly useful for discovering phosphorylation-dependent protein-protein interaction networks from cell lysates or transfected cells.biosynthetic pathway that converts phosphoenolpyruvate into nhpSer inside the cell (Figure 2) to increase bioavailability of nhpSer for subsequent encoding into a protein, providing > 40-fold improvement in nhpSer-protein production and enabling scalability of expressions (Zhu et al., 2023).This "PermaPhos" expression system provided us with sufficient quantities of nhpSer-containing proteins to confirm that nhpSer mimics pSer function in several cases where aspartate/glutamate do not (Zhu et al., 2023).We outline here the general workflow for expressing a control protein (super folder GFP, sfGFP) containing nhpSer at site N150, and confirming correct encoding.Strategies to adopt this protocol for biologically relevant proteins are discussed.
Figure 1.Biosynthesis of serine in E. coli.In this protocol, a ∆serC mutant expression host is used as it lacks the ability to biosynthesize pSer amino acid, which would compete with nhpSer encoding.SerB is also overexpressed to hydrolyze any free pSer amino acid that might enter the cell from the media or be formed by promiscuous transaminases that can substitute for SerC function.For GCE systems that encode authentic phosphoserine, ∆serB expression hosts are used in order to build up intracellular pSer concentrations.FrbA, B, C, D, and E proteins.In this protocol, the pCDF-Frb-v1 plasmid expresses five Frb enzymes that, along with an unknown transaminase in E. coli, convert the central metabolite phosphoenolpyruvate into nhpSer, which is then encoded into target proteins using the GCE machinery.prevent biosynthesis of phosphoserine, which would compete with nhpSer for incorporation into proteins (Figure 1).This strain contains Release Factor 1 (RF1), the protein responsible for terminating translation at TAG amber codons, so truncated protein will be produced along with full-length nhpSer-protein.To avoid copurification of truncated protein with full-length protein, C-terminal purification tags are recommended.For proteins that self-assemble into homo-multimers (dimers, trimers, etc.), purification can be challenging due to the possible co-purification of truncated forms that are incorporated as subunits in the assembly.If using Nterminal purification/solubilization tags, additional purifications steps may be needed to remove truncated protein species.See General note 1 for more discussion.2. DH10b (Thermo Fisher, catalog number: EC0113).This strain can be used for faithful propagation of plasmids and for cloning needs when users wish to clone their genes of interest into the pRBC plasmid (see Plasmids below).Do not use for protein expression.Though we have not explicitly tested all of them, other classical cloning strains of E. coli can be used including NEB 10-beta (New England BioLabs, catalog number: C3019H), DH5α (e.g., Thermo Fisher, catalog number: 18258012), NEB 5-alpha (New England BioLabs, catalog number: C2987H), or TOP10 (Thermo Fisher, catalog number: C404010).

Plasmids
1. pERM2-nhpSer (Addgene, #201922): machinery plasmid for nhpSer incorporation, kanamycin resistance with pUC origin of replication.This plasmid expresses the same tRNA synthetase and EF-Tu used for pSer incorporation (Rogerson et al., 2015).The amber codon suppressing Sep-tRNACUA is v2.0 developed by Chin and colleagues to minimize mis-aminoacylation (Zhang et al., 2017).The Sep-tRNA and tRNA-synthetase are constitutively expressed via lpp and GlnS promoters, respectively.The EF-Tu is under control of an IPTG inducible tac promoter.Also constitutively expressed via an OXB20 promoter is the E. coli serB protein to further eliminate (hydrolyze) free pSer amino acid that may come from the media or produced by promiscuous transaminases.2. pCDF-Frb-v1 (Addgene, #201923): expresses Streptomyces rubellomurinus FrbA, FrbB, FrbC, FrbD, and FrbE enzymes required for the biosynthesis of nhpSer.See Zhu et al. (2023) for a detailed description of this biosynthetic pathway, which is also summarized in Figure 2.Each Frb protein is expressed under the control of an engineered T7 transcriptional promoter variant and so all Frb enzyme are expressed by the addition of IPTG.This plasmid confers spectinomycin resistance and contains the CloDF origin of replication.This plasmid is large (~11 kb) and contains repetitive elements; while we have not observed instability of this plasmid, given its large size it is good practice to minimize propagations.We recommend that when you receive the DH10b cells with the pCDF-Frb-v1 plasmid from Addgene, grow up a few individual colonies overnight in liquid media [e.g., 2× YT media supplemented with spectinomycin at 100 μg/mL (see Recipes)] and make frozen glycerol stocks of the cells for long-term storage.Glycerol stocks can be made by mixing 600 μL of overnight culture with 400 μL of sterile 50% (v/v) glycerol.Place culture tube(s) in -80 °C freezer for storage.These stocks serve as a permanent, long-term source of plasmid, which can be prepared by inoculating cultures with cells from a glob of the frozen glycerol stock.Do not thaw the glycerol stock(s) once frozen.n/a 250 mL Total n/a 500 mL Mix by placing a suitable magnetic stir bar in a 500 mL graduated cylinder and add 250 mL of water to graduated cylinder.While stirring, pour pure glycerol to the 500 mL mark on graduated cylinder.Stir for 5 min and then transfer to a 0.5 L bottle.Sterilize by autoclaving on liquid setting.

10% (v/v) glycerol (1 L)
Reagent Final concentration Quantity Glycerol (100%) 10% (v/v) 100 mL H2O n/a 900 mL Total n/a 1 L Mix by placing a suitable magnetic stir bar in a 1,000 mL graduated cylinder and add 900 mL of water to graduated cylinder.While stirring, pour pure glycerol to the 1,000 mL mark on graduated cylinder.Stir for 5 min and then transfer to a 1 L bottle.Sterilize by autoclaving on liquid setting.

SOC media, 50 mL
Reagent Final concentration Quantity 2× YT media n/a 49 mL 1 M MgSO4 10 mM 0.5 mL 40% (w/v) α-D-glucose 0.4% (w/v) or ~20 mM 0.5 mL Total n/a 50 mL a. 1 M MgSO4 can be made by mixing 12.3 g of MgSO4•7H2O in water up to 50 mL of total volume.Adjust mass of MgSO4 if using the salt with different hydration status.b. 40% (w/v) α-D-glucose can be made by mixing 20 g of α-D-glucose with water up to 50 mL of total volume.Mix thoroughly until glucose is dissolved.Gentle heating in a microwave may facilitate dissolution of glucose.c.Sterilize MgSO4, glucose, and 2× YT solutions individually by autoclaving.Allow each component to cool to room temperature and mix as indicated above.Maintain sterility while adding components together.

Procedure
In Part A, you first prepare electro-competent BL21(DE3) ∆serC cells before expressing protein in Part B. These electro-competent cells need to be highly efficient for expressions to be successful (see Troubleshooting 2).The process outlined below is sufficient to produce enough of electro-competent cells for ~20-30 transformations and expressions.In a small Styrofoam box, a pan, or dish-like container, add dry ice pellets and pour enough ethanol (or isopropanol) to cover the bed of dry ice.Place an empty 1 mL pipette tip rack on the bed of dry ice; the base of the pipette rack should be touching the ethanol but should not be submerged, such that when you place a 0.6 mL Eppendorf tube in the rack, the bottom half of the Eppendorf tube is suspended in the ethanol bath while the cap of the tube stays well above the ethanol.p.When the final centrifuge run is complete, pour off or aspirate the supernatant.Resuspend the cell pellet gently with ~0.3 mL of 10% (v/v) glycerol into an even suspension and transfer to a sterile 15 mL conical tube.To resuspend the cells, you can use a 1,000 μL pipettor equipped with a pipette tip that has had ~3 mm cut off the end with a sterile razor blade.The reason for cutting off the end of the pipette tip is to widen the hole at the end to minimize shearing forces on your cells as you are pipetting them up and down to resuspend.q.Once resuspended into an even cell suspension, measure OD600.

A. Preparation of electro-competent BL21(DE3) ∆serC cells
Note: You will need make a ~1:500 dilution of the cells to get an accurate OD600 reading, e.g., 998 μL of water + 2 μL of cell suspension.Measure the OD600 of these diluted cells and then calculate OD600 of your actual cell suspension based on the dilution factor.For example, if your OD600 reading of your 1:500 dilution = 0.5, then the cell suspension is 0.5 × 500 = 250.r.The OD600 of your resuspended cells should be between 200 and 300 to achieve sufficient competency for triple plasmid transformations and expressions.If the OD600 is above 300, dilute the cells with cold 10% glycerol so that the final density lies between 200 and 300.If the OD600 is below 200, then centrifuge the cells again and resuspend in a smaller volume of 10% (v/v) glycerol, e.g., 0.1 mL instead of 0.3 mL, re-measure OD600, and adjust volumes as needed.s.Pipette ~35 μL of cell suspension into a 0.6 mL Eppendorf tube and quickly place tube in the dryice/ethanol bath rack to freeze cells.The bottom of the Eppendorf tube should be submerged in the dry ice/ethanol bath, but do not let the cap of the tube come in contact with the ethanol; the alcohol will wick its way into the tube, ruining the cells.t.Once cells are frozen (~2 min in the dry ice/ethanol bath), remove tube from bath, wipe the tube free of ethanol with a Kimwipe, and place the tube with frozen cells in a cryogenic freezer box containing dry ice.u.Repeat until all cells have been aliquoted and frozen.Once all aliquots are frozen and in the box with dry ice, place box at -80 °C for long-term storage.You should obtain ~20-30 aliquots of competent cells from this procedure.v.When done, place dry-ice/ethanol bath in a fume hood to allow dry ice to sublime.The ethanol/isopropanol can be poured into a bottle for re-use.Caution: the ethanol/isopropanol will offgas CO2 for many hours, so if you pour it into a bottle be sure to leave cap lose/off to allow gas to escape, ensuring pressure does not build up inside the bottle.

B. Expression of protein with site-specifically encoded nhpSer
• Fresh triple plasmid transformations must be performed for each expression.Never freeze expression cells containing plasmids; if expression cells are frozen with expression plasmids in them, the cells will grow in the presence of the correct antibiotics, but they will not express protein.Do not sequentially transform plasmids.• To standardize this protocol in your lab, first run control expressions with sfGFP wt and sfGFP-150TAG to verify you can achieve the reference expression benchmarks (see Table 1 below) and ensure that nhpSer encoding at the TAG site is functioning as expected with a model protein.The protocol below outlines the protocol for a standard 50 mL test expression to encode nhpSer into sfGFP at position N150 (i.e., sfGFP-150TAG), as well as wild-type sfGFP.Pointers on how to scale up to a 1 L expression, as is often used for target proteins, are also included.1. Day 1: transformations a. Prepare two LB/agar plates, one for sfGFP-wt and one for sfGFP-150TAG expressions, with antibiotics as follows: i. Sterilize LB/agar as described above.After autoclaving, allow to cool sufficiently to touch while still remaining liquid.
ii. Pour 50 mL of LB/agar into a sterile 50 mL conical tube.Add 35 μL each of ampicillin, kanamycin, and spectinomycin stock solutions.Mix thoroughly and pour ~20-25 mL into each 100 mm plate.
iii.Allow tubes to chill on ice for 5-10 min.d.Thaw two aliquots of electrocompetent BL21(DE3) ∆serC cells.Cells can be thawed rapidly with the warmth of your fingers, but immediately place tube on ice once thawed.e. Transfer 30 μL of electrocompetent BL21(DE3) ∆serC cells to each tube with plasmids and gently mix cells with plasmids by pipetting up and down 2-3 times.Cells should not appear stringy or behave like "snot," which would indicate cells have lysed.f.Transfer the "sfGFP-WT" electro-competent cell/plasmid mix into a pre-chilled electro-cuvette.Wipe metal electrode plates dry with a Kimwipe.Gently flick the cuvette to ensure cells are sitting in the bottom and stretch the full width of the cuvette.g.Place electro-cuvette into the electroporator and electroporate according to instrument instructions for 1 mM cuvettes (e.g., 1,500 V for the Eppendorf Eporator).

Note: The time constant should be 4-6 ms. If arc'ing occurs, discard cells and cuvette. Arc'ing occurs when DNA and/or cells have too much salt. Re-attempt electroporation by diluting a fresh cell/plasmid mix with equal volume of cold MQ water.
h. Immediately add 1 mL of SOC media to electro-cuvette and resuspend cells.Transfer cells to a sterile 1.7 mL Eppendorf tube.i. Repeat steps f-h for the "sfGFP-150TAG" electro-competent cell/plasmid mix.j.Place both Eppendorf tubes in a shaker (250 rpm) and recover cells at 37 °C for 90 min.
Note: Electro-cuvettes can be reused at least 10 times if washed shortly after use.Wash cuvettes as follows while cells are recovering: i. MQ H2O.ii.0.1 M HCl (dilute acid helps to break down residual DNA in the cuvette).
iii.MQ H2O.iv.70% (v/v) ethanol.v. Let dry upside down on a Kimwipe.Once dry, store with cap to maintain sterility.k.After 90 min of recovery, plate all the cells from the "sfGFP-WT" recovery culture onto the LB/agar/Amp/Kan/Spec plate labeled "sfGFP-WT." To plate all the transformed cells, you cannot put the entire 1 mL of recovery culture on the agar plate as this is too much liquid and it will not dry properly.Instead, pellet the cells by centrifugation at 3,000× g for 3 min.Remove the top 900 μL of media and gently resuspend the pellet in the remaining ~100 μL.Spread this cell suspension evenly onto the agar plate.l.Repeat plating process for the "sfGFP-150TAG" culture.m.Let plates dry with lid partially open for ~20 min near a flame (maintaining sterility) and then incubate plates upside down overnight (~16 h) at 37 °C.

Day 2: expressions
• This protocol is intentionally written to avoid overnight liquid starter cultures that would reach stationary phase.Rather, in one day, you will use freshly grown colonies on an agar plate to grow a starter liquid culture in the morning, after which you will inoculate expression culture in the early afternoon.By the end of the day the cultures will be induced and allowed to express for the next 20-24 h.Having a large number (> 500) of colonies on each LB/agar plate is necessary to execute this one-day expression protocol.• All liquid cultures must contain 70 μg/mL ampicillin, 35 μg/mL kanamycin, and 70 μg/mL spectinomycin.• Important: Baffled culture flasks are critical to ensure adequate aeration of expression cultures.a. Remove LB/agar plates from 37 °C incubator.There should be several hundred or thousands of colonies (examples of successful triple plasmid transformations are shown in Figure 3).ii.For small-scale (e.g., 50 mL) sfGFP control expressions, prepare two starter cultures by adding 5 mL of starter culture media (with ampicillin, kanamycin, and spectinomycin antibiotics) to 14 mL sterile culture tubes.iii.To inoculate these 5 mL cultures, scrape a "glob" of cells constituting several dozen to hundreds of colonies from overnight LB/agar plate with a sterile pipette tip, shake the glob off into the culture media, and break apart by gentle pipetting.Enough cells should be transferred to the 5 mL starter culture such that it is visibly turbid upon inoculation.iv.For larger scale expressions (e.g., > 1 L), prepare 50 mL of starter culture media (with antibiotics) in a baffled 250 mL culture flask.Transfer 5 mL of starter culture media onto the LB/agar plate containing transformed cells.Gently scrape all colonies with a sterile glass spreader to resuspend them into the liquid media.Transfer media with suspended cells into the 250 mL baffled culture flask containing ~45 mL of starter culture media.Add 1-2 drops of antifoam.c.Grow starter cultures at 37 °C with shaking at 250 rpm for ~3-4 h until OD600 > 1. Do not grow overnight.d.While starter cultures are growing, prepare expression media: i.For each 50 mL sfGFP test expression, add 5 mL of (room temperature) sterile 10× potassium phosphate buffer to 45 mL of 1.1× TB media in a 250 mL baffled culture flask.Add antibiotics and ~1-2 drops of antifoam.
ii.For 1 L expressions, add 100 mL of sterile 10× potassium phosphate buffer to 900 mL of 1.1× TB media in a 2.8 L baffled Fernbach flask.Add antibiotics and ~5-6 drops of antifoam.e.After ~3-4 h of starter culture growth, inoculate expression media as follows: i.For 50 mL of sfGFP test expressions, add 1 mL of the sfGFP-WT starter culture to 50 mL of TB expression media (in a baffled 250 mL culture flask).Repeat for the sfGFP-150TAG starter culture.
ii.For a 1 L expression, add 10-20 mL of starter culture to each baffled Fernbach flask containing 1 L of TB expression media.f.Grow expression cultures at 37 °C with shaking (200-250 rpm) until OD600 = 1.0.This should take approximately 3-5 h.g.When cultures reach OD600 = 1, add IPTG to a final concentration of 0.5 mM.i.For 50 mL cultures, this corresponds to 50 μL of 0.5 M IPTG stock solution.
ii.For 1 L cultures, this corresponds to 1 mL of 0.5 M IPTG stock solution.h.Reduce temperature to 30 °C for sfGFP control expression cultures.For other target proteins, temperature can be reduced to as low as 20 °C.The Frb biosynthetic pathway functions optimally between 20 and 30 °C.Expression temperatures below 20 °C may be possible but expression times will need to be extended and overall yields will be diminished.Do not express target proteins above 30 °C since the Frb biosynthetic pathway does not function above this temperature.i.For 50 mL expression cultures, add 1-2 more drops of antifoam.For 1 L expressions, add 5-6 more drops of antifoam.j.Continue shaking cultures at 200-250 rpm for 20-24 h at the desired temperature (between 20 and 30 °C; optimal for sfGFP is 30 °C).During this expression period, the density of the culture (OD600) should increase significantly (up to 20).

Day 3: Evaluating expression results and harvesting cells sfGFP control protein expression analysis
Approximately 20-24 h after induction, measure OD600 and fluorescence of both sfGFP and sfGFP- 150TAG cultures using a fluorometer.Since sfGFP chromophore formation requires synthesis of fulllength protein, fluorescence of whole cells provides a convenient strategy to evaluate the efficiency of sfGFP TAG codon suppression, and therefore nhpSer incorporation.Fluorescence can be measured with any fluorimeter capable of detecting sfGFP fluorescence (ex/em: 488/510 nm).Diluting the culture 1:10 to 1:100 in a buffer (e.g., 100 μL of culture + 1,900 μL of PBS for a 1:20 dilution) prior to fluorescence measurements may be necessary to obtain a signal within the linear range of the fluorometer.

Test expression benchmarks:
In general, the sfGFP-150TAG culture fluorescence should be approximately 20%-30% that of the sfGFP wild-type culture, corresponding to 50-150 mg of sfGFP-150nhpSer per liter of culture (Table 1).The OD600 values will vary depending on target protein.Normal values will range from ~10 to 20 for sfGFP expressions.Final OD600 values below 5 are indicative of poor cell growth or toxicity due to target protein expression.

Harvesting cells:
Harvest cells by centrifugation at 5,000× g for 15 min at 4 °C.Pour off culture supernatant and resuspend cells in an appropriate buffer.
• The choice of buffer depends on the protein of interest, the downstream purification strategy, and the application, and should be determined by the user.• Adding a cryoprotectant [e.g., 10% (v/v) glycerol] to this buffer can help minimize adverse effects associated with freezing sensitive or unstable proteins.Cells can be flash frozen in liquid nitrogen and stored at -80 °C or you can proceed with purification.• For His6-tagged proteins to be purified via TALON resin, a recommended resuspension/lysis buffer would be 50 mM Tris pH 7.5, 500 mM NaCl, 10% (v/v) glycerol, 5 mM imidazole.Phosphatase inhibitors are not required since nhpSer is not hydrolyzed by phosphatases.

Validation of protocol
For each expression, it is important to confirm faithful incorporation of nhpSer into the target protein.Several methods can be used to evaluate the phosphorylation status of the target protein.
1. Phos-tag electrophoresis: Phos-tag gel electrophoresis is a modified form of SDS-PAGE, in which phosphorylated proteins migrate with attenuated electrophoretic mobility compared to their nonphosphorylated counterparts.The degree to which protein migration is attenuated increases with sequential addition of phosphoryl groups, allowing one to distinguish between no, single-, and multi-pSer containing  ) and the sfGFP-150nhpSer protein was produced using the protocol described here.Because they migrate differently on Phos-tag gels, nonphosphorylated, pSer-containing, and nhpSer-containing proteins can be resolved from one another, and the fraction of each form can be easily evaluated in each sample.In this example, the sfGFP-150pSer protein

1 . 10 Published
Day 1 a.Prepare LB/agar plate (no antibiotics) and cool to room temperature to solidify.b.Streak out BL21(DE3) ∆serC cells onto LB/agar plate from a small glob of cells taken from a frozen glycerol stock.c.Place LB/agar plate with streaked cells upside down in a static 37 °C incubator and grow overnight (14-20 h).2.Day 2a.After overnight growth, place LB/agar plate with BL21(DE3) ∆serC cells in a refrigerator until the end of the day.b.Prepare and autoclave 1 L of 2× YT media in a 2.8 L baffled Fernbach flask.c.Prepare 1 L of 10% (v/v) glycerol and sterilize by autoclaving along with the following: i. 500 mL graduated cylinder.ii. 2 × 0.5 L centrifuge bottles (or bottles suitable for centrifuging 1 L of culture at 4,000× g).Centrifuge bottles should be thoroughly cleaned to remove any residual DNA or cells that could contaminate electrocompetent cells prior to autoclaving.iii.A box each of 1,000, 100, and 10 μL pipette tips.iv.Approximately fifty 0.6 mL Eppendorf tubes.d.After autoclaving, place the 10% (v/v) glycerol solution in a refrigerator or cold room to chill overnight.e.At the end of the day, add 5 mL of 2× YT media to a 14 mL culture tube and inoculate with a single colony of BL21(DE3) ∆serC cells grown the prior night on the LB/agar plate.Grow liquid culture at 37 °C overnight with shaking at 250 rpm. 3. Day 3 a.Inoculate 1 L of 2× YT media (in a 2.8 L baffled Fernbach flask) with the 5 mL of overnight starter culture.Add ~5-6 drops of anti-foam to the culture flask.Excessive foaming of the media caused by the flask baffles with inhibit air exchange and result in slower cell growth.b.Grow the 1 L culture at 37 °C with shaking at 200-250 rpm until OD600 ~0.4.This should take approximately 2-3 h.Do not let culture grow above OD600 ~0.5.It is a good idea to check the OD600 approximately 1.5 h after inoculation to verify if cells are growing.c.While cells are growing, pre-chill centrifuge and rotor to 4 °C and place centrifuge bottle(s) and graduated cylinder in refrigerator to chill as well.Cite as: Zhu, P. et al. (2023).Biosynthesis and Genetic Encoding of Non-hydrolyzable Phosphoserine into Recombinant Proteins in Escherichia Coli.Bio-protocol 13(21): e4861.DOI: 10.21769/BioProtoc.4861.Once cells reach OD600 ~0.4,immediately place culture flask in an ice/water bath for 15 min.Mix culture occasionally to ensure efficient cooling.Note: From this point forward, work quickly and keep cells ice-cold at all times.Never let cells warm and work in a cold room if necessary.Maintain sterility.e. Pour chilled culture into two sterilized 0.5 L centrifuge bottles and centrifuge cells at 4,000× g for 15 min at 4 °C.f.Pour supernatant off without disturbing cell pellet.Wipe any residual media supernatant around the lip of the centrifuge bottle if necessary.Place centrifuge bottle with cell pellet immediately on ice.g.Resuspend each cell pellet with 250 mL each of ice-cold 10% (v/v) glycerol solution (can be measured with the sterile graduated cylinder) by gentle swirling on ice.Be gentle with the cells, and DO NOT VORTEX.Keep cells cold at all times.h.Once cells are evenly resuspended in 10% (v/v) glycerol, combine into one 0.5 L centrifuge bottle and centrifuge again at 4,000× g for 15 min at 4 °C.i.After centrifuging, pour supernatant off without disturbing cell pellet.centrifuge bottle with cell pellet on ice.j.Measure 250 mL of 10% (v/v) ice-cold glycerol in the same pre-chilled graduated cylinder.Resuspend cell pellet with this 250 mL of ice-cold glycerol solution by gentle swirling on ice.Again, DO NOT VORTEX and keep cells and centrifuge bottle cold at all times during the resuspension process.k.Once cells are evenly resuspended in 10% (v/v) glycerol, centrifuge again at 4,000× g for 15 min at 4 °C.l.Pour supernatant off without disturbing cell pellet.Place centrifuge bottle with cell pellet on ice.m.Resuspend cell pellet with 30 mL of ice-cold glycerol solution by gentle swirling on ice.Again, DO NOT VORTEX and keep cells and centrifuge bottle cold at all times during the resuspension process.Transfer resuspended cells to a sterile 50 mL conical tube and top off conical tube to 50 mL total volume with ice cold 10% (v/v) glycerol.n.Centrifuge again at 4,000× g for 15 min at 4 °C.o.During this final centrifuge run, prepare a dry ice/ethanol bath as follows:
Cite as: Zhu, P. et al. (2023).Biosynthesis and Genetic Encoding of Non-hydrolyzable Phosphoserine into Recombinant Proteins in Escherichia Coli.Bio-protocol 13(21): e4861.DOI: 10.21769/BioProtoc.4861.15 Published: Nov 5, 2023proteins(Kinoshita et al., 2006).Serendipitously, proteins with nhpSer migrate slower on Phos-tag electrophoresis than the same proteins with authentic pSer, allowing easy discrimination between the two [for examples, please see(Zhu et al., 2023) as well as Figure4below].By measuring the density of the nonphosphorylated vs. pSer vs. nhpSer protein bands, one can estimate the percentage of expressed/purified protein that contains nhpSer.Important to note is that wild-type (non-phosphorylated) protein must be run side-by-side with the phosphorylated protein to observe relative shifts in electrophoretic mobility.Similarly, it is also helpful to express your target proteins with pSer as well so that you can confirm if the electrophoretic shifts of your nhpSer-containing protein are due to nhpSer encoding and not pSer [see our previous protocol for expressing proteins with pSer:(Zhu et al., 2022)].Other advantages of Phos-tag electrophoresis include the ability to evaluate multiple samples at once, requiring only a standard SDS-PAGE electrophoresis setup, and being economical.Do not run molecular weight markers on Phos-tag gels, as they often contain EDTA, which is not compatible with Phos-tag.See Figure4below for an example of SDS-PAGE and Phos-tag gels of nhpSer and pSer containing sfGFP.Note: The attenuated electrophoretic mobility of sfGFP-150pser/nhpSer proteins in Phos-tag gels are particularly pronounced and easily discernable; however, the degree to which other phosphorylated proteins migrate slower than their non-phosphorylated counterparts depends on the protein and the site of encoding.If you do not see a convincing electrophoretic shift between your phosphorylated and non-phosphorylated proteins, consider optimizing the Phos-tag electrophoresis procedure to improve resolving power by (i) increasing concentration of the Phos-tag acrylamide reagent from 25 or 50 μM to 100 μM or higher, (ii) decreasing the amount of protein loaded for narrower bands and enhanced resolution between similarly migrating protein bands, (iii) decrease the total acrylamide concentration [e.g., from 15% to 10% (w/v) acrylamide] so that target proteins bands travel at least half-way through the gel thereby increasing separation, and (iv) run sfGFP WT/150nhpSer control proteins alongside the proteins of interest to ensure the Phos-tag gel is run correctly.If still no shift is observed on Phos-tag electrophoresis, you will have to confirm incorporation via mass spectrometry (see below).

Figure 4 .
Figure 4. Assessing nhpSer encoding into sfGFP by Phos-tag gel electrophoresis.SDS-PAGE (top) and Phos-tag (bottom) gels of purified WT sfGFP, sfGFP-150pSer, and sfGFP-150nhpSer proteins.The sfGFP-150pSer protein was produced following our prior protocol (Zhu et al., 2022) and the sfGFP-150nhpSer protein was produced using the protocol described here.Because they migrate differently on Phos-tag gels, nonphosphorylated, pSer-containing, and nhpSer-containing proteins can be resolved from one another, and the fraction of each form can be easily evaluated in each sample.In this example, the sfGFP-150pSer protein

Cite as: Zhu, P. et al. (2023). Biosynthesis and Genetic Encoding of Non-hydrolyzable Phosphoserine into Recombinant Proteins in Escherichia Coli. Bio-protocol 13(21): e4861. DOI: 10.21769/BioProtoc.4861. 6 Published: Nov 5, 2023 a
. After mixing reagents thoroughly, autoclave on standard liquid setting to sterilize.Note the agar will not go into solution until autoclaved.b.After autoclaving, gently swirl the bottle to ensure melted agar is evenly mixed.After mixing reagents thoroughly, autoclave on the standard liquid setting to sterilize.b.After autoclaving, allow to cool to room temperature before use.

9. Ampicillin stock (10 mL) Reagent Final concentration Quantity
Sterilize by autoclaving on liquid setting.b.Do not mix TB phosphate buffer (below) until all solutions are cool and immediately before use.The masses provided for the potassium phosphate salts are for anhydrous formulations.Hydrate forms of these salts can be used, but masses must be adjusted to maintain indicated molarity.b.Sterilize by autoclaving on liquid setting.c.Do not mix TB phosphate buffer to 1.1× TB media until all solutions are sterilized and cooled to room temperature, and immediately before use.Sterilize by filtering with a 0.2 μm syringe-end filter.b.Store in 1 mL aliquots at -20 °C.

Table 1 . Benchmarks for sfGFP-WT and 150TAG protein expression yields, based on culture fluorescence Fluorescence of culture # mg of sfGFP per liter culture
# The range indicated in parenthesis is considered normal depending on the day and reagent preparation.Fluorescence values reported here were obtained on a hand-held PicoFluor fluorometer (Turner Biosystems) by diluting cells directly from the culture into PBS (1:20).Fluorescence values are arbitrary and will depend on the fluorometer used.It is important that the relative ratio of sfGFP-150TAG and sfGFP-WT culture fluorescence is consistent with the above values.For reference, background cultures not expressing sfGFP have auto-fluorescence values of ~150-250.*Yield of sfGFP in milligrams per liter was determined using a fluorescence standard curve of purified sfGFP.